Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation

Because of its relative inaccessibility, inflammatory cell extravasation within the airway circulation in vivo has been difficult to investigate in real time. A new method has been established using intravital microscopy in the anesthetized rat to visualize leukocytes in superficial postcapillary venules of the trachea. This technique has been validated using local superfusion of lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Basal leukocyte rolling velocity (55.4 +/- 9.3 microm/s) and adhesion (1.4 +/- 0.3 cells/100 microm) were monitored in postcapillary venules (33.9 +/- 1.3 microm diameter). At all time points up to 90 min, these parameters were unaltered in control rats (n = 7). In contrast, vessels exposed to 1 microg/ml of LPS (n = 6) exhibited a 57% reduction in leukocyte rolling velocity and an increase in the number of adherent cells (4.7 +/- 1 cells/100 microm, P < 0.05). Superfusion with 0.1 microM of FMLP (n = 6) also resulted in a 45% reduction in rolling velocity and an increase in adherent cells (4 +/- 0.7 cells/100 microm, P < 0.05). Histological evaluation confirmed local stimulus-induced leukocyte extravasation. These results demonstrate leukocyte recruitment in the airway microvasculature and provide an important new method to study airway inflammation in real time.     

Stretching the timescale of intravital imaging in tumors

Since the time it was pioneered in 1992, intravital imaging of tumors at cellular resolution has offered us the extremely important opportunity of “seeing biology”. However, until now, most studies were monitoring tumor cell behavior in the same animal over short times, requiring the combining of acquired data into a hypothesis via statistical analysis. In the last several months, different groups have independently developed techniques to extend the time scale of intravital imaging to several days. This improvement allows one to address the connection between tumor cell behavior and the microenvironment which surrounds them. We can now assess dynamics of the cell-cell interactions in tumors, analyze tumor cell fate and changes in the tumor extracellular matrix which accompany tumor progression.     

A method of intravital study of pulmonary microcirculation in closed-chest cats

The methods of intravital study of the cat lungs have been updated. A device has been designed for the investigation of microvessels in extended lung areas of spontaneously breathing closed-chest cats. The principles for quantitative analysis of microcirculatory lung parameters have been elaborated. These new methods allow the study of important natural phenomena of the lung microvascular functions.     

Stretching the timescale of intravital imaging in tumors

Since the time it was pioneered in 1992, intravital imaging of tumors at cellular resolution has offered us the extremely important opportunity of “seeing biology.” However, until now, most studies were monitoring tumor cell behavior in the same animal over short times, requiring the combining of acquired data into a hypothesis via statistical analysis. In the last year, different groups have independently developed techniques to extend the time scale of intravital imaging to several days. This improvement allows one to address the connection between tumor cell behavior and the microenvironment which surrounds them. We can now assess dynamics of the cell-cell interactions in tumors, analyze tumor cell fate and changes in the tumor extracellular matrix which accompany tumor progression.Key words: intravital, multiphoton, spinning disc, microenvironment, second harmonic generation, mammary imaging window, dorsal skinfold chamber, photoswitchingIntravital imaging of tumors at cellular resolution offers insight into the physiology of cells in vivo in real time. The first published study which included injectable dyes to monitor tumor metastasis inside the embryo was done by the group of Groom.¹ Some years later, Farina,² and then Naumov,³ and co-workers, used GFP-labeled tumor cells to study tumors by confocal scanning microscopy. Soon after, Brown,⁴ and Wang,⁵ and co-workers, introduced two-photon microscopes into their studies.Until recently, single cell-resolved intravital imaging in tumors commonly involved recording movies 4D (3D through time) with one or two channels, collecting data via multiphoton microscopy from one region at a time.^6–8 The inner side of the orthotopic tumor is exposed by making a small incision in the skin and skin folding. This technique, termed ‘skin-flap’, allows for several hours of imaging in one animal. Data from several animals are combined into the final result averaging measurements as well as differences in tumor preparation, animal condition and genotype. Some low-resolution studies have proposed a reversible flap⁹ on the tumor tissue implanted several days earlier. However, visualized areas were not the same at each of the timepoints. Also, as skin flaps were opened repeatedly, they were potentially influencing the microenvironment by surgery-related immune/inflamatory-responses. In addition, several groups have been using a dorsal skinfold chamber¹⁰ in which the tumor is grown ectopically, in the space between the skin and glass coverslip on the back of the mouse. This preparation could be used for either low resolution measurements over several days, or short-term measurements at cellular resolution.In the last few months, several studies have included techniques which extend the time-scale of intravital imaging in tumors from hours to days (

Noninvasive intravital imaging of thymocyte dynamics in medaka

In vivo imaging of thymocytes has not been accomplished due to their localization deep within opaque body and high susceptibility to surgical stress. To overcome these problems, medaka is useful because of transparency and ex-uterine development. We report the noninvasive detection of thymocytes in transgenic medaka that express fluorescent protein under the control of immature-lymphocyte-specific rag1. We show that lymphoid progenitor cells colonize the thymus primordium in an anterior-to-posterior orientation-specific manner, revealing that extrathymic anterior components guide prevascular thymus colonization. We also show that developing thymocytes acquire "random walk motility" along with the expression of Ag receptors and coreceptors, suggesting that thymocyte walking is initiated at the developmental stage for repertoire selection. Thus, transgenic medaka enables real-time intravital imaging of thymocytes without surgical invasion.     

Comparative study of intravital microcirculation and hemorheological changes following burn injury of different severity in rats

It was shown in experiments on rats that burn injury is followed by microcirculatory disturbances, hemoconcentration and increasing blood viscosity that is especially pronounced in the vessels with low blood pressure. The microcirculatory changes in the mesentery correlated with the in vitro investigated dynamic viscosity and blood composition. The disturbances were more pronounced after severe burn followed by a mortal shock than after moderate burn without fatal consequences. This investigation confirms great importance of hemorheological changes and microcirculatory disturbances in the early period of burn disease.     

Static and dynamic errors in particle tracking microrheology

Particle tracking techniques are often used to assess the local mechanical properties of cells and biological fluids. The extracted trajectories are exploited to compute the mean-squared displacement that characterizes the dynamics of the probe particles. Limited spatial resolution and statistical uncertainty are the limiting factors that alter the accuracy of the mean-squared displacement estimation. We precisely quantified the effect of localization errors in the determination of the mean-squared displacement by separating the sources of these errors into two separate contributions. A "static error" arises in the position measurements of immobilized particles. A "dynamic error" comes from the particle motion during the finite exposure time that is required for visualization. We calculated the propagation of these errors on the mean-squared displacement. We examined the impact of our error analysis on theoretical model fluids used in biorheology. These theoretical predictions were verified for purely viscous fluids using simulations and a multiple-particle tracking technique performed with video microscopy. We showed that the static contribution can be confidently corrected in dynamics studies by using static experiments performed at a similar noise-to-signal ratio. This groundwork allowed us to achieve higher resolution in the mean-squared displacement, and thus to increase the accuracy of microrheology studies.     

SWIP—a stabilized window for intravital imaging of the murine pancreas

Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.     

以消散场成像和单微粒跟踪技术揭示GLUT4囊泡和分泌囊泡的不同动态过程

葡萄糖转运子蛋白4(glucose transporter 4,GLUT4)在维持体内葡萄糖动态平衡的过程中起着至关重要的作用。GLUT4贮存囊泡(GLUT4 storage vesicle,GSV)和神经内分泌细胞中的分泌囊泡含有许多相同的蛋白。研究证明这些蛋白调节了分泌囊泡的胞内转运过程,但是GLUT4囊泡和分泌囊泡是否具有相同的胞内动态过程还未阐明。文章以3T3-L1纤维原细胞中的GSV和神经内分泌细胞PC12细胞中的分泌囊泡:致密核心大囊泡(large dense core vesicle,LDCV)为研究对象,使用消散场显微成像技术和单微粒跟踪技术直观观察了活体细胞内单个GSV和LDCV的三维运动轨迹。通过以适当方程拟合单个囊泡的均方位移曲线,发现两种囊泡都具有三种运动模式。定量分析显示作自由扩散运动和方向性扩散运动的GSV数量明显多于LDCV。对比GSV和LDCV的三维扩散系数,发现GSV的扩散系数中值为7.2×10-4μm2/s,而LDCV的扩散系数中值仅为1.94×10-4μm2/s。这一结果说明GSV的活动性远大于LDCV,提示GSV的胞内转运过程涉及不同的分子机制。     

Targeted particle tracking in computational models of human carotid bifurcations

A significant and largely unsolved problem of computational fluid dynamics (CFD) simulation of flow in anatomically relevant geometries is that very few calculated pathlines pass through regions of complex flow. This in turn limits the ability of CFD-based simulations of imaging techniques (such as MRI) to correctly predict in vivo performance. In this work, I present two methods designed to overcome this filling problem, firstly, by releasing additional particles from areas of the flow inlet that lead directly to the complex flow region ("preferential seeding") and, secondly, by tracking particles both "downstream" and "upstream" from seed points within the complex flow region itself. I use the human carotid bifurcation as an example of complex blood flow that is of great clinical interest. Both idealized and healthy volunteer geometries are investigated. With uniform seeding in the inlet plane (in the common carotid artery (CCA)) of an idealized bifurcation geometry, approximately half the particles passed through the internal carotid artery (ICA) and half through the external carotid artery. However, of those particles entering the ICA, only 16% passed directly through the carotid bulb region. Preferential seeding from selected regions of the CCA was able to increase this figure to 47%. In the second method, seeding of particles within the carotid bulb region itself led to a very high proportion (97%) of pathlines running from CCA to ICA. Seeding of particles in the bulb plane of three healthy volunteer carotid bifurcation geometries led to much better filling of the bulb regions than by particles seeded at the inlet alone. In all cases, visualization of the origin and behavior of recirculating particles led to useful insights into the complex flow patterns. Both seeding methods produced significant improvements in filling the carotid bulb region with particle tracks compared with uniform seeding at the inlet and led to an improved understanding of the complex flow patterns. The methods described may be combined and are generally applicable to CFD studies of fluid and gas flow and are, therefore, of relevance in hemodynamics, respiratory mechanics, and medical imaging science.     

Study of hydrodynamics in microcarrier culture spinner vessels: A particle tracking velocimetry approach

Three-dimensional particle tracking velocimetry (3-D PTV), a modern, quantitative, visualization tool, has been applied to the characterization of the flow field in the impeller region of cell culture reactor vessels. The experimental system used here is a 250-mL microcarrier spinner vessel. The studies were conducted at three different agitation rates, 90, 150, and 210 rpm, corresponding to healthy, mildly damaging, and severely damaging shear intensities, respectively. The flow can be classified into three regions: a predominantly tangential (azimuthal) flow generated by the impeller; a trailing vortex region coming off the impeller tip; and a converging flow region close to the center of the vessel. The latter two are the regions of highest velocity gradients. Energy dissipation rates due to mean velocity gradients were also calculated to characterize the impeller stream. Local specific energy dissipation rates > 10,000 erg/(cm(3)sec) . have been measured. It is proposed that the critical regions for microcarrier culture damage due to impeller hydrodynamics are the trailing vortex region and the high energy converging flow region. Graphical representation of the mean velocity flow fields and the distribution of energy dissipation rates in the impeller region are also presented here. The merits of using the dissipation function (measure of specific energy dissipation rate) as a possible scale-up parameter are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Lens density tracking in mice by Scheimpflug imaging

Scheimpflug imaging has recently been established for in vivo imaging of the anterior eye segment and quantitative determination of lens transparency in the mouse. This enables more effective investigations of cataract formation with the mouse model, including longitudinal studies. In order to enable recognition of disease-associated irregularities, we performed Scheimpflug measurements with the common laboratory inbred lines C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129/SvJ in a period between 2 and 12 months of age. C57BL/6J mice showed lowest mean lens densities during the test period. Progressive cortical lens opacification was generally observed, with the earliest onset in C57BBL/6J, C3HeB/FeJ, and 129/SvJ, between 2 and 6 months after birth. Moreover, lenses of these inbred lines developed nuclear opacities. Calculated mean lens density significantly increased between 6 and 12 months of age in all inbred strains except 129/SvJ. Lens densities (and the corresponding standard deviations) of FVB/NCrl and 129/SvJ increased most likely because of differences in the genetic background. Albinism as confounder might be excluded since the albino Balb/cByJ mice are more similar to the C57BL/6J or C3Heb/FeJ mice. We further identified strain-specific anterior lens opacities (C57BL/6J) and cloudy corneal lesions (C57BL/6J, FVB/NCrl, and BALB/cByJ) at later stages. In conclusion, our results indicate that there are lifelong opacification processes in the mouse lens. The highest lens transparency and a dark coat color, which prevents interference from light reflections, make mice with the C57BL/6J background most suitable for cataract research by Scheimpflug imaging. We show that lens densitometry by Scheimpflug imaging in mouse eyes can resolve differences of less than 1 %, making it possible to detect differences in cataract development in different mouse strains, even if they are small.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.